Measurement Method Using Oxidase

ABSTRACT

A method for measuring a target object in a sample by using an oxidase, wherein the influence of dissolved oxygen in the sample can be corrected, is provided. The method comprises: obtaining measurement values by causing the target object in the sample to react with the oxidase under different conditions of two or more types; and performing a correction based on the obtained two or more measurement values and a correction method preliminarily set so as to correct the influence of dissolved oxygen in the sample.

TECHNICAL FIELD

The present invention relates to a measurement method using an oxidase,and to a biosensor and a measurement device used in the measurementmethod.

BACKGROUND ART

Practical application of enzyme sensors has been advanced particularly,as compared with other biosensors. For example, enzyme sensors formeasuring glucose, lactic acid, cholesterol, lactose, uric acid, urea,and amino acids are used in medical measurement and in the foodindustry. An enzyme sensor performs a quantitative analysis of ananalyte by reducing an electron acceptor (mediator) with electronsgenerated by reaction between an enzyme and a target object to bemeasured (substrate) contained in a sample solution that is a liquid,and electrochemically measuring an oxidation-reduction degree of theelectron acceptor. In the case where blood is the sample solution,however, there is the problem that an accurate assay cannot be performedby a biosensor using an oxidation-reduction enzyme, due to the influenceof dissolved oxygen. Particularly, an enzyme sensor utilizing GOD(glucose oxidase) is often used in measurement upon a pre-meal insulininjection and evaluation of hypoglycemia. If a higher concentration ofglucose than the actual level is indicated due to the influence ofdissolved oxygen, this could lead to excessive administration ofinsulin, and the hypoglycemia would remain undetected. Therefore, abiosensor is in demand that is not influenced by dissolved oxygen evenif it is used with respect to a sample that could possibly containdissolved oxygen, such as blood.

As a technique for avoiding the influence of dissolved oxygen, a methodusing a third electrode (JP 10(1998)-282038 A, JP 2000-065778 A), and anenzyme sensor using GDH (glucose dehydrogenase) (JP 2007-163499 A), etc.have been developed. On the other hand, it has been pointed out that newcapital investment is needed in the case of the method using a thirdelectrode, and that as to the method using GDH, enzyme costs of GDH arehigher than that of GOD, and the method is likely to be influenced bysaccharides such as maltose that hinder the measurement.

SUMMARY OF THE INVENTION Problem to be Solved by the Invention

to cope with these problems, a measurement method using anoxidation-reduction enzyme biosensor, with which capital investment canbe reduced and enzyme costs can be decreased, has been desired. Thepresent invention provides a method for measuring a target object in asample using an oxidase, wherein the influence by dissolved oxygen inthe sample can be corrected.

Means to Solve the Problem

The present invention relates to a method for measuring a target objectin a sample, the method including: obtaining measurement values byreacting a target object in a sample with an oxidase under differentconditions of two or more types; and performing a correction based onthe obtained two or more measurement values and a correction methodpreliminarily set so as to correct influences of dissolved oxygen in thesample.

The present invention in another aspect relates to a biosensorincluding: two or more independent electrode systems each of whichcomprises a working electrode and a counter electrode provided on asubstrate; and oxidase-containing reagents provided respectively on thetwo or more electrode systems, the oxidase-containing reagents beingcapable of measuring a single target object in a single sample underdifferent conditions. Further, the present invention in still anotheraspect relates to a measurement device for measuring a target object,the measurement device including: a sensor section for obtaininginformation about measurement results obtained from reactions between atarget object in a sample and an oxidase under different conditions oftwo or more types; a memory section for storing a correction method; anarithmetic section for selecting the correction method stored in thememory section based on the information obtained by the sensor section,and calculating a corrected value of the information by the correctionmethod; and a display section for displaying the corrected value.

Effects of the Invention

With the measurement method of the present invention, even in thepresence of dissolved oxygen, a concentration of a target object can bedetermined with the influence of dissolved oxygen being corrected withuse of an oxidase. Further, the present invention preferably makes itpossible to correct the influence of dissolved oxygen without using anenzyme other than an oxidase, for example, GDH. Further, the presentinvention preferably makes it possible to correct the influence ofdissolved oxygen without using a third electrode.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating a configuration of anembodiment of a biosensor according to the present invention.

FIG. 2 is a block diagram illustrating a configuration of one embodimentof a measurement device according to the present invention.

FIG. 3 is a flowchart showing an action of one embodiment of themeasurement device according to the present invention.

FIG. 4 is a graph showing results of examples.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on knowledge that, in measurements usingoxidase electrode systems, corrections based on results in two types ofreaction systems that differ from each other in reaction conditions (forexample, the oxygen amount, the type of mediator, etc.) can reduce theinfluence of dissolved oxygen.

More specifically, the present invention relates to a method formeasuring a target object in a sample using an oxidase, the measurementmethod including:

obtaining measurement values by causing the target object in the sampleto react with the oxidase under different conditions of two or moretypes; and performing a correction based on the obtained two or moremeasurement values and a correction method preliminarily set so as tocorrect the influence of dissolved oxygen in the sample (hereinafterthis method is also referred to as “the measurement method of thepresent invention”). The measurement method of the present inventionenables measurement in which influences of dissolved oxygen in a samplecan be corrected.

The mechanism of predicting an amount of dissolved oxygen based onmeasurement values obtained by measurement under different conditions oftwo or more types is considered to be as follows. When an oxidase enzymeis used, the concentration of dissolved oxygen influences theoxygen-mediator reaction (see the scheme shown below). It is presumedthat variation of a magnitude of this influence depending on differencesin the reaction conditions makes an output time course different.However, the interpretation of the present invention may not be limitedto the interpretation based on this mechanism.

In the present specification, “target object” refers to an object thatis to be measured by the method of the present invention and thatbecomes a substrate of an oxidase. Examples of the same include, but arenot limited to, glucose, lactic acid, bilirubin, cholesterol, andascorbic acid. Therefore, as to the oxidase that is to react with atarget object, any oxidase having the target object as a substratethereof can be used in the present invention. Examples of such anoxidase include, but are not limited to, glucose oxidase, lactateoxidase, bilirubin oxidase, cholesterol oxidase, and ascorbic acidoxidase. Further, examples of the oxidase that can be used in themeasurement method of the present invention include, but are not limitedto, uric acid oxidase, peroxidase, sulfurous acid oxidase, amineoxidase, lysyl oxidase, lysine oxidase, amino acid oxidase, diamineoxidase, pyridoxine phosphate oxidase, protein-lysine 6-oxidase,acyl-CoA oxidase, alcohol oxidase, choline oxidase, pyruvic acidoxidase, sarcosine oxidase, tyramine oxidase, and glycerophosphateoxidase. Substrates of these may be target objects.

In the present specification, the “sample” refers to, unless otherwiseprovided particularly, a composition or a mixture used in themeasurement method of the present invention in which a target objectexists or can possibly exist. Examples of the sample include liquidcontaining a target object or liquid that can generate a target object,for example, biological samples such as blood, plasma, serum, urine, andbody fluid.

In the present specification, “causing a target object in a sample toreact with an oxidase” refers to bringing a sample into contact with anoxidase so as to cause a target object to react with the oxidase. Areagent containing an oxidase may be of a dry type or of a wet type, butfrom the viewpoint of simplification, a sample and an oxidase arepreferably brought into contact with each other on a biosensor.“Measurement values” are, for example, electric current values in thecase where electrode systems are used, or absorbances or transmittancesin the case where optical detection is performed, but they are notparticularly limited as long as they are measurement values obtained bydetection about reaction between a target object and an oxidase. Fromthe viewpoint of simplification, “causing a target object in a sample toreact with an oxidase” is preferably performed in a biosensor providedwith electrode systems. In the case where electrode systems are used, amediator is preferably contained in a reaction system.

In the present specification, “different conditions of two or moretypes” refers to different conditions of at least two types under whicha reaction is caused to occur, the conditions of at least two typesbeing different regarding at least one of conditions of reaction betweena target object and an oxidase, so that measurement values of at leasttwo types are obtained. The different reaction conditions are notparticularly limited, but examples of the same include a type and/oramount of an oxidase, a reaction temperature, a reaction time, a typeand/or amount of a mediator, a type and/or amount of a hydrophilicpolymer, a type and/or amount of a surfactant, and a type and/or amountof a saccharide.

In the specification, examples of the “measurement value” includeresults detected about reaction between a target object and an oxidase,and more specifically include electric current values, resistancevalues, absorbances, transmittances, and values obtained from these (forexample, concentrations calculated with use of finite differences,areas, and calibration curves).

[Correction Method]

A correction method can be set preliminarily by preliminarily obtainingmeasurement values from a plurality of samples having differentdissolved oxygen concentrations under predetermined reaction conditionsof two or more types. The following can be suggested, for example: aquotient or a difference of respective measurement values A and B ofdifferent reaction systems A and B is related with a concentration ofdissolved oxygen, and a correction method is set based on it. It shouldbe noted that in the present specification, a measurement valuecorrected by the measurement method of the present invention is referredto as “corrected value”.

One embodiment of the correction method is, for example, correctionusing a quotient of measurement values A and B (B/A or A/B) that areobtained from reaction systems A and B, respectively. One example is amethod of obtaining a corrected concentration by multiplying aconcentration A obtained from a measurement value A with a quotient B/Aof measurement values. It should be noted that regarding the obtainmentof a concentration A from a measurement value A, it may be, asconventionally, performed by using calibration curves, etc. Thiscorrection method is based on the following knowledge: that influencesby dissolved oxygen on an oxidase reaction system depend on an amount ofa reagent such as an oxidase in the reaction system; and that inreaction systems A and B that are different in reaction conditions of areagent containing an oxidase, correction can be achieved bymultiplication with a quotient (B/A or A/B) of measurement values A andB. It should be noted that in the present embodiment, from the viewpointof improving correction accuracy further, multiplication with acorrection coefficient c in addition to the quotient of measurementvalues is preferred. This constant depends on predetermined reactionconditions, and it can be set easily by preliminarily performingexperiments under the predetermined reaction conditions. Therefore, apreferable style of correction in the present embodiment is correctionof multiplying a concentration A obtained from a measurement value Awith a quotient B/A of measurement values and a correction coefficientc. The constant c may be 1.

Another embodiment of the correction method suggested herein is, forexample, as follows: a concentration (or range thereof) of dissolvedoxygen is related with a quotient or a difference of measurement valuesA and B based on preliminarily obtained date, andexecution/non-execution of correction and contents of the correction(e.g., an amount of correction, a correction coefficient, etc.)corresponding to the concentration or range of dissolved oxygen are set.

[Biosensor]

In the measurement method of the present invention, reaction between atarget object and an oxidase may be performed in a biosensor includingelectrode systems each of which has a working electrode and a counterelectrode, and reagent layers containing an oxidase, the layers beingprovided on the electrode systems. It should be noted that the reagentlayer contains a mediator generally.

Examples of the mediator usable in the biosensor include, but are notlimited to, potassium ferricyanide, sodium ferricyanide, p-benzoquinoneand derivatives of the same, phenazine methosulfate and derivatives ofthe same, indophenol and derivatives of the same, potassiumβ-naphthoquinone-4-sulfonate, 2,6-dichlorophenol indophenol, methyleneblue, nitrotetrazolium blue, ferrocene and derivatives of the same,osmium complex, ruthenium complex, NAD+, NADP+, and pyrroloquinolinequinone (PQQ).

The reagent layers of the biosensor may further contain a hydrophilicpolymer, a surfactant, and a saccharide. As these components furthercontained therein, those used in conventional biosensors of oxidaseelectrode types can be used.

In an exemplary embodiment, the biosensor used in the measurement methodof the present invention is equipped with two or more independentelectrode systems. Such a biosensor enables measurement whereininfluences by dissolved oxygen are corrected, with only one measurementoperation.

Therefore, the present invention in still another aspect relates to abiosensor used in the measurement method of the present invention, thebiosensor comprising: two or more independent electrode systems each ofwhich has a working electrode and a counter electrode provided on asubstrate; and an oxidase, and a mediator as required, the oxidase andthe mediator being provided on the two or more electrode systems. Asdescribed above, it is preferable that on the two or more independentelectrode systems, reagent layers having different conditions areformed, respectively. More specifically, the reagent layers arepreferably reagent layers that differ in at least one of a type and/oramount of an oxidase, a type and/or amount of a mediator, a type and/oramount of a hydrophilic polymer, a type and/or amount of a surfactant, atype and/or amount of a saccharide, and the like. Therefore, the presentinvention in still another aspect relates to a biosensor comprising: twoor more independent electrode systems each of which has a workingelectrode and a counter electrode provided on a substrate; andoxidase-containing reagents provided respectively on the two or moreelectrode systems, the oxidase-containing reagents being capable ofmeasuring a single target object in a single sample under differentconditions.

An embodiment of a biosensor used in a measurement method according tothe present invention is explained below with reference to FIG. 1. FIG.1 is an exemplary schematic view illustrating a configuration of anembodiment of a biosensor according to the present invention. Thebiosensor of the present embodiment comprises: two independent electrodesystems each of which has a working electrode 2 and a counter electrode3 that are provided on a substrate 1; reagent layers 4 including anoxidase and a mediator, the reagent layers being provided on theelectrode systems, respectively; a flow path 5; and air vents 6. Thebiosensor of the present invention, however, is not limited to thisembodiment. In the biosensor shown in FIG. 1, a sample introduced intothe flow path 5 flows toward the air vents 6, branches at a branchingpoint, and is brought into contact with the two reagent layers 4provided on the electrodes 2 and 3, where reaction occurs. The resultsof the reaction are detected from the electrodes 2 and 3.

[Measurement Device]

The present invention in still another aspect relates to a measurementdevice that comprises: a sensor section for obtaining information aboutmeasurement results of two or more types obtained from reactions betweena target object in a sample and an oxidase under different conditions oftwo or more types; a memory section for storing a correction method; anarithmetic section for selecting the correction method stored in thememory section based on the information obtained by the sensor section,and calculating a corrected value of the information by the correctionmethod; and a display section for displaying the corrected value. Themeasurement device of the present invention can be used in themeasurement method of the present invention.

An embodiment of a measurement device according to the present inventionis explained below with reference to FIG. 2. FIG. 2 is an exemplaryblock diagram illustrating a configuration of an embodiment of ameasurement device according to the present invention. In FIG. 1, themeasurement device of the present embodiment includes a control section21, a display section 22, a sensor section 23, an arithmetic section 24,a memory section 25, and a power source section 26. The measurementdevice of the present invention may have a configuration in which a partor all of these sections including the control section, the displaysection, the sensor section, the arithmetic section, and the memorysection are integrally provided, or alternatively, may have aconfiguration in which they are individually provided.

The sensor section 23 is a section to which a biosensor is connectableand from which, after the biosensor is connected (attached),information, for example, electric current values, is obtained.Therefore, the sensor section may include a voltage application portion,an electric current value determination portion, and the like. Thememory section 25 is intended, for example, to store a correctionmethod, a correction coefficient, etc., and to store data and the liketo be used in selection of a correction method.

The arithmetic section 24 is intended to set and/or select a correctionmethod stored in the memory section 25 based on information obtained bythe sensor section 23, and to calculate a corrected value of theinformation corrected by the correction method. The selection of acorrection method can be performed by, for example, comparinginformation obtained by the sensor section 23 with the following datastored in the memory section 25: data that relate measurement values anddissolved oxygen concentrations with each other; and/or data that relatemeasurement values and contents of correction such as correctioncoefficients with each other. In the present invention, “informationobtained by the sensor section 23” may include measurement valuesdetected by the biosensor in the sensor section, and data based on themeasurement values (for example, statistic values such as average valuesand dispersion, a difference between two measurement values, or thelike). The calculation of the corrected value may be decidedappropriately by the selected correction method, and may be carried outby, for example, multiplying a measurement value with a correctioncoefficient. Further, the arithmetic section 24 can calculate aconcentration of a target object, and the like, using calibration curvesand the like, in the case where the corrected value is an electriccurrent value or the like.

The display section 22 is intended to display results of arithmeticoperations by the arithmetic section 24, for example, correctedconcentrations, etc., predicted values regarding dissolved oxygen in asample, a correction coefficient, and the like. The display 22 is formedwith, for example, a liquid crystal display device.

The power supply section 26 is intended to supply electric power to thecontrol section 21, the display section 22, the sensor section 23, thearithmetic section 24, and the memory section 25. Further, in the casewhere the measurement device is a measurement device for a biosensorhaving electrodes, the power supply section 26 is used for applying avoltage across a working electrode and a counter electrode of thebiosensor, and determining an amount of electrons transferred betweenthe working electrode and the mediator. Further, the control section 21is intended to control the display section 22, the sensor section 23,the arithmetic section 24, the memory section 25, and the power supplysection 26.

Referring to the flowchart of FIG. 3, the following explains a methodfor measuring a target object with use of the measurement device shownin FIG. 2, taking as an example a case where the target object isglucose and the biosensor is a biosensor having electrode systems.Needless to say, however, the following explains merely one example, andthe present invention is not limited to this.

First, a biosensor is attached to the sensor section 23 of themeasurement device. Then, a sample such as blood is introduced into thebiosensor, so as to react with a target object in the sample with anoxidase. Then, in the sensor section 23, a voltage is applied across aworking electrode and a counter electrode of the biosensor, and aresponse current at that time is measured, whereby a measurement value(data) is obtained (S31). Whether or not measurement values (data) underdifferent conditions of two or more types have been obtained isdetermined by the control section 21 (S32). In the case where thecontrol section 21 determines that measurement values under differentconditions of two or more types have not been obtained, the attempt ofobtaining data is repeated again (S31). In the case where the controlsection 21 determines that measurement values under different conditionsof two or more types have been obtained, the arithmetic section 24performs a correction method determination operation (S33). In thecorrection method determination operation, the correction method isdecided and set with reference to, for example, data thus obtained and acorrection method preliminarily stored in the memory section 24. Then,data are corrected by the correction method thus set (S34), and acorrected value, or a concentration calculated based on the correctedvalue, etc., is displayed on the display section 22 (S35).

EXAMPLE [Measurement of Glucose by Two GOD Electrode System (A and B)]

Using a biosensor provided with two GOD electrode systems (A and B),samples 1 to 3 were subjected to measurement operations. Reagentscontained in reagent layers in the electrode systems A and B are asshown in Table 1 below. It should be noted that the unit (U) in Table 1is defined to be an amount of enzyme that is capable of altering 1micromole (μmol) of a substrate per one minute under optimal conditions(at a temperature of 30° C., and at a degree of acidity at which thechemical reaction proceeds most). Further, concentrations of glucose anddissolved oxygen in each sample are as shown in Table 2 below. Samples 1to 3 are prepared using venous blood. The concentration of glucose wasadjusted by adding glucose, and the concentration of dissolved oxygenwas adjusted by shaking a tube containing the venous blood. It should benoted that the method for measuring the concentration of glucose and theconcentration of dissolved oxygen is as follows.

TABLE 1 Component Mediator Reagent layer Enzyme (Ruthenium Surfactant ofbiosensor (GOD) complex) (Sucrose laurate) System A 2.0 U 20 μg 0.2 ngSystem B 0.5 U 20 μg 0.2 ng

TABLE 2 Composition Glucose Dissolved oxygen Sample 1 336 mg/dL 30 mmHgSample 2 336 mg/dL 70 mmHg Sample 3 336 mg/dL 110 mmHg 

[Measurement by Biosensor]

Using the biosensor provided with the above-described reagent layers(systems A and B), the samples 1 to 3 were measured (n=8). It should benoted that, regarding the measurement device, GA-1150 (trade name,produced by ARKRAY Inc.) was used as a glucose concentration meter, andABL5 (trade name, produced by Radiometer) was used as a dissolved oxygenconcentration meter. The results of the measurement are shown in Table 3below.

TABLE 3 n 1 2 3 4 5 6 7 8 Sample 1 System A 11.23716 11.04659 11.6576312.89393 10.70419 10.20777 10.82521 10.70419 System B 10.69173 10.1401410.34778 9.572999 10.59562 10.32179 9.706331 10.59562 Sample 2 System A10.44701 10.16902 9.675613 9.055767 10.94827 10.53663 9.667293 10.94827System B 9.952365 9.894451 9.670695 8.949746 9.817386 9.247258 9.844429.817386 Sample 3 System A 9.311172 10.01974 10.06494 9.952288 9.47045410.29546 9.837839 9.470454 System B 9.378793 10.20714 10.6195 9.85548110.06338 9.586116 10.76204 10.06338 Unit: μA

[Relating of Measurement Value and Dissolved Oxygen Concentration, andDecision of Correction Method]

Based on the data shown in Table 3 above, the measurement values of thesystems A and B and concentrations of dissolved oxygen in the sampleswere related with one another, and a correction method as shown belowwas decided. It should be noted that in the present example, thecorrection coefficient mentioned below is 1.04.

Corrected Value

=(sample concentration determined from data of system A)×(measurementvalue of system B/measurement value of system A)×correction coefficient

The results of correction by the above-described correction method areshown in Table 4 below and FIG. 4. The table 4 below shows averagevalues of non-corrected values (concentrations obtained by referring toonly measurement value of the system A and applying the same to acalibration curve), relative standard deviations (S.D.), coefficients ofvariation (C.V.), alienations (%) from a reference value (336 mg/dL),and corresponding values after correction. FIG. 4 is a graph showing therelationship between concentrations of dissolved oxygen in samples, andalienations (%) from the reference value before correction and thoseafter the correction.

TABLE 4 Non-corrected value (Data obtained from system A) Correctedvalue Alienation Alienation from from Dissolved Average S.D. referenceAverage S.D. reference oxygen (mg/dL) (mg/dL) C.V. value (%) (mg/dL)(mg/dL) C.V. value (%) Sample 1 30 mmHg 366.63 26.51 7.2% 9.12 350.1314.43 4.1% 4.21 Sample 2 70 mmHg 335.00 21.72 6.5% −0.30 330.22 12.063.7% −1.72 Sample 3 110 mmHg  322.77 11.23 3.5% −3.94 344.74 16.10 4.7%2.60

As shown in Table 4 and FIG. 4, measurement values with the influence bydissolved oxygen being reduced were obtained by the above-describedcorrection method.

INDUSTRIAL APPLICABILITY

The present invention is useful in the medical field, the life sciencefield, and the biological research field.

The invention may be embodied in other forms without departing from thespirit or essential characteristics thereof. The embodiments disclosedin this application are to be considered in all respects as illustrativeand not limiting. The scope of the invention is indicated by theappended claims rather than by the foregoing description, and allchanges which come within the meaning and range of equivalency of theclaims are intended to be embraced therein.

1-5. (canceled)
 6. A measurement device comprising: a sensor section forobtaining information about measurement results obtained from reactionsbetween a target object in a sample and an oxidase under differentconditions of two or more types; a memory section for storing acorrection method; an arithmetic section for selecting the correctionmethod stored in the memory section based on the information obtained bythe sensor section, and calculating a corrected value of the informationby the correction method; and a display section for displaying thecorrected value.
 7. The measurement device according to claim 6, whereinthe target object is selected from the group consisting of glucose,lactic acid, bilirubin, cholesterol and ascorbic acid.
 8. Themeasurement device according to claim 6, wherein the target object isglucose.
 9. The measurement device according to claim 6, wherein theoxidase is selected from the group consisting of glucose oxidase,lactate oxidase, bilirubin oxidase, cholesterol oxidase, ascorbic acidoxidase, uric acid oxidase, peroxidase, sulfurous acid oxidase, amineoxidase, lysyl oxidase, lysine oxidase, amino acid oxidase, diamineoxidase, pyridoxine phosphate oxidase, protein-lysine 6-oxidase,acyl-CoA oxidase, alcohol oxidase, choline oxidase, pyruvic acidoxidase, sarcosine oxidase, tyramine oxidase and glycerophosphateoxidase.
 10. The measurement device according to claim 6, wherein theoxidase is glucose oxidase.